This proposal will continue to study the physiology of mouse dendritic cells (DCs), to explain 3 cardinal features: potency in controlling cell-mediated immnunity, positioning in vivo to capture antigens and migrate to T cell areas of lymphoid organs, and priming of CD4 and CD8 protective immune responses. In culture, we will probe the efficiency with which DCs capture antigens and form immunogenic MHC-peptide complexes. In vivo, we will dissect the distinctive features of Langerhans cells, the DCs of the epidermis, and subsets of immature and mature DCs. Our specific aims will address 4 emerging areas of DC biology. 1) Antigen capture and TCR ligand formation via DEC-205: This multilectin receptor, through its cytosolic domain, uniquely recycles through MHC II positive lysosomal compartments and enhances presentation. 2) Costimulation of naive T cells: DCs stimulate TCR transgenic T cells more efficiently (>30x) than activated B cells, even when the two cell types have comparable levels of surface MHC II peptide complexes. We will address costimulation and formation of immunological synapses by each APC, as well as in vivo homing and helper T cell differentiation. 3) Langerhans cell biology: The high efficiency with which these DCs home to T cell areas, and their capacity to elicit MHC class I responses, will be addressed. 4) Genetic targeting of DCs. To unravel the biology of DCs in the periphery and in T cell areas, the genes for mouse DC-SIGN, Langerin and DC-LAMP will be isolated. Monoclonal antibodies will be prepared to study these molecules that are selectively expressed on immature and/or mature DCs. The ovalburnin antigen and GFP sequences will be knocked into these new genes, to study the distribution and immunologic consequences of gene expression in DCs. Together these aims will provide reagents, methods and data needed to understand DC physiology and their use in immune therapy of chronic infections and malignancy.